Core Technology
TBD
Key Features
| Technical Aspect/Parameters | GFP Expression Marker System | Conventional Approach |
|---|---|---|
| Signal source | Used Aequorea victoria green fluorescent protein as the visible marker. | Used reporter systems such as β-galactosidase, firefly luciferase, or bacterial luciferase. |
| Need for added compounds | Patent stated detection required near-UV or blue light and no exogenously added compounds. | Patent stated these methods could require added substrates or cofactors. |
| Gene expression readout | A regulatory element was operatively linked to GFP so fluorescence indicated expression. | Not specified. |
| Localization method | A protein of interest was fused to GFP so fluorescence showed location in the cell. | Older marker methods described in the patent often required added substrates, cofactors, or fixed preparations. |
| Cell selection method | In the claimed method, cells expressing GFP were selected to select cells expressing the paired protein of interest. | Not specified. |
| Host range described | The patent described bacterial, yeast, fungal, insect, nematode, plant, and animal cells. | Not specified. |
| Stimulus and detection use | Promoter-controlled GFP indicated heavy metals, stress responses, pollutants, or certain target molecules described in the patent. | Not specified. |
Technical Specifications
| Technical Specification | Reported in Patent |
|---|---|
| Source protein | Aequorea victoria green fluorescent protein (GFP) |
| Protein length | 238 amino acids |
| Main excitation peak | 395 nm |
| Minor excitation peak | 470 nm |
| Peak emission | 509 nm |
| Emission shoulder | 540 nm |
| Detection mode | Fluorescence under near-UV or blue light |
| Substrates/cofactors | Not required |
| Gene products | Not required |
| Construct | pGFP10.1 |
| Coding change | Codon 80, CAG→CGG |
| Amino acid change | Gln→Arg |
| Reported effect of mutation | No detectable change in spectral properties |
| Model organisms | E. coli and C. elegans |
| ATCC deposit | pGFP10.1, Accession 75547 |
Applications
- Monitored gene expression in living cells by linking a regulatory element to GFP.
- Localized a protein of interest in a cell by expressing a GFP fusion protein.
- Selected cells that expressed a protein of interest by selecting cells that fluoresced.
- Separated fluorescent cells with fluorescence-activated cell sorting.
- Tracked the effects of external stimuli on a regulatory element through changes in fluorescence.
- Determined tissue-specific activity of a DNA sequence in a subject.
- Indicated gene expression in cells or subjects through visible fluorescence.
- Detected heavy metals in a solution using a promoter activated by heavy metal exposure.
- Detected pollutants in a solution using heavy-metal-responsive or stress-protein promoters.
- Detected target bacteria or cell types using bacteriophages or viruses that carried the GFP gene.
Development Status
Validation to date included the original patent demonstrations in E. coli and C. elegans, and the technology later developed into a widely used research tool for live-cell imaging, gene expression reporting, and protein localization. After the original patent period, GFP and related fluorescent proteins were further engineered into broader imaging toolsets and were recognized in 2008 by the Nobel Prize in Chemistry for the discovery and development of GFP.
Categories
- Impact Areas:
- Health & Medicine, Pollution & Waste Systems, Research & Innovation
- Technology Areas:
- Biotechnology
